PDZ domain from Dishevelled -- a specificity study

Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt β-catenin pathway at the expense of non-canonical Wnt signaling.     

Identification of tripeptides recognized by the PDZ domain of Dishevelled

The development of inhibitors of Dishevelled (Dvl) PDZ protein–protein interactions attracts attention due to a possible application in drug discovery and development. Using nuclear magnetic resonance (NMR) spectroscopy, we found that a tripeptide VVV binds to the PDZ domain of Dvl, which is a key component involved in Wnt signaling. Using a computational approach calculating the binding free energy of the complexes of the Dvl PDZ domain and each of the tripeptides VXV (X: any amino acid residue except Pro), we found that a tripeptide VWV had the highest binding affinity. Consistent with the computational result, experimental results showed that the binding of the tripeptide VWV to the Dvl PDZ domain was stronger than that of the tripeptide VVV. The binding affinity of the tripeptide VWV was comparable to that of the organic molecule NSC668036, which was the first identified Dvl PDZ inhibitor. The three-dimensional structure of the complex Dvl1 PDZ/VWV was determined to investigate the role of the energetically favorable W(?1) residue in binding. These interactions were also explored by using molecular dynamic simulation and the molecular mechanics Poisson–Boltzmann surface area method. Taken together, these two tripeptides may be used as modulators of Wnt signaling or as a scaffold to optimize an antagonist for targeting Dvl1 PDZ protein–protein interaction.     

Paxillin binding to a conserved sequence motif in the alpha 4 integrin cytoplasmic domain

alpha(4)beta(1) integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other beta(1) integrins. Paxillin, a signaling adapter protein, binds tightly to the alpha(4) cytoplasmic domain and is implicated in alpha(4) integrin signaling. We now report the mapping of a paxillin-binding site in the alpha(4) cytoplasmic domain and an assessment of its role in the alpha(4) tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu(983)-Tyr(991)) within the alpha(4) cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr(991) and Glu(983) as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length alpha(4) tail sequence. Y991A or E983A substitution disrupted the interaction of alpha(4) integrins with paxillin. These same two point mutations reversed the effects of the alpha(4) tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all alpha(4) integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of alpha(4) integrins.     

A C-terminal PDZ binding domain modulates the function and localization of Kv1.3 channels

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms.     

Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain

PDZ domains are protein-protein interaction modules found in hundreds of human proteins. Their binding reactions are sensitive to variations in salt and pH but the basis of the respective dependence has not been clear. We investigated the binding reaction between PSD-95 PDZ3 and a peptide corresponding to a native ligand with protein engineering in conjunction with stopped-flow and equilibrium fluorimetry and found that the two conserved residues Arg-318 and His-372 were responsible for the salt and pH dependencies, respectively. The basis of the salt-dependent variation of the affinity was explored by mutating all charged residues in and around the peptide-binding pocket. Arg-318 was found to be crucial, as mutation to alanine obliterated the effect of chloride on the binding constants. The direct interaction of chloride with Arg-318 was demonstrated by time-resolved urea denaturation experiments, where the Arg-318 --> Ala mutant was less stabilized by addition of chloride as compared with wild-type PDZ3. We also demonstrated that protonation of His-372 was responsible for the increase of the equilibrium dissociation constant at low pH. Both chloride concentration and pH (during ischemia) vary in the postsynaptic density, where PSD-95 is present, and the physiological buffer conditions may thus modulate the interaction between PSD-95 and its ligands through binding of chloride and protons to the "molecular switches" Arg-318 and His-372, respectively.     

Peptide binding to the PDZ3 domain by conformational selection

The PDZ domains, a large family of peptide recognition proteins, bind to the C‐terminal segment of membrane ion channels and receptors thereby mediating their localization. The peptide binding process is not known in detail and seems to differ among different PDZ domains. For the third PDZ domain of the synaptic protein PSD‐95 (PDZ3), a lock‐and‐key mechanism was postulated on the basis of the almost perfect overlap of the crystal structures in the presence and absence of its peptide ligand. Here, peptide binding to PDZ3 is investigated by explicit solvent molecular dynamics (MD) simulations (for a total of 1.3 μs) and the cut‐based free energy profile method for determining free energy barriers and basins. The free energy landscape of apo PDZ3 indicates that there are multiple basins within the native state. These basins differ by the relative orientation of the α2 helix and β2 strand, the two secondary structure elements that make up the peptide binding site. Only the structure with the smallest aperture of the binding site is populated in the MD simulations of the complex whose analysis reveals that the peptide ligand binds to PDZ3 by selecting one of three conformations. Thus, the dynamical information obtained by the atomistic simulations increment the static, that is, partial, picture of the PDZ3 binding mechanism based on the X‐ray crystallography data. Importantly, the simulation results show for the first time that conformational selection is a possible mechanism of peptide binding by PDZ domains in general. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

The RNA binding domain of the hantaan virus N protein maps to a central, conserved region

The nucleocapsid (N) protein of hantaviruses encapsidates both viral genomic and antigenomic RNAs, although only the genomic viral RNA (vRNA) is packaged into virions. To define the domain within the Hantaan virus (HTNV) N protein that mediates these interactions, 14 N- and C-terminal deletion constructs were cloned into a bacterial expression vector, expressed, and purified to homogeneity. Each protein was examined for its ability to bind the HTNV S segment vRNA with filter binding and gel electrophoretic mobility shift assays. These studies mapped a minimal region within the HTNV N protein (amino acids 175 to 217) that bound vRNA. Sequence alignments made from several hantavirus N protein sequences showed that the region identified has a 58% identity and an 86% similarity among these amino acid sequences. Two peptides corresponding to amino acids 175 to 196 (N1) and 197 to 218 (N2) were synthesized. The RNA binding of each peptide was measured by filter binding and competition analysis. Three oligoribonucleotides were used to measure binding affinity and assess specificity. The N2 peptide contained the major RNA binding determinants, while the N1 peptide, when mixed with N2, contributed to the specificity of vRNA recognition.     

Angiomotin belongs to a novel protein family with conserved coiled-coil and PDZ binding domains

Angiomotin has previously been identified in a yeast two-hybrid screen by its ability to bind to angiostatin, an inhibitor of novel formation of blood vessels (angiogenesis). Angiomotin mediates the inhibitory effect of angiostatin on endothelial cell migration and tube formation in vitro. Here we report that two human protein sequences, of which one is novel and one has been cloned previously, are similar to angiomotin and are members of a novel protein family, which we propose to call motins. These two genes have been named angiomotin-like 1 (amotl1) and angiomotin-like 2 (amotl2). We have cloned mouse angiomotin and identified amotl1 and amotl2 homologs in mice. The alignment of the amino acid sequences encoded by these six sequences spans 455 residues of which 64% was conserved in all six proteins. Sequence analysis showed that these sequences all share putative coiled-coil domains and PDZ-binding motifs. Sequence information from GenBank indicate that motins can be found in several species including the frog Xenopus laevis, the pufferfish Fugu rubripes and the nematode Caenorhabditis elegans. Further phylogenetic analysis indicates that amotl2 is an evolutionary outgroup in relation to angiomotin and amotl1. Northern blot analysis shows distinct expression patterns for each motin in various mouse tissues.     

Regulation of c-Src by binding to the PDZ domain of AF-6

c-Src is a tightly regulated non-receptor tyrosine kinase. We describe the C-terminus of c-Src as a ligand for a PDZ (postsynaptic density 95, PSD-95; discs large, Dlg; zonula occludens-1, ZO-1) domain. The C-terminal residue Leu of c-Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c-Src regulation in cells. As a candidate PDZ protein, we analysed AF-6, a junctional adhesion protein. The AF-6 PDZ domain restricts the number of c-Src substrates, whereas knockdown of AF-6 has the opposite effect. Binding of c-Src to the AF-6 PDZ domain interferes with phosphorylation of c-Src at Tyr527 by the C-terminal kinase, and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src kinase. Unphosphorylated Tyr527 allows binding of c-Src to AF-6. This can be overcome by overexpression of CSK or strong activation of c-Src. c-Src is recruited by AF-6 to cell-cell contact sites, suggesting that c-Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c-Src regulation by interaction with a PDZ protein.     

Association of Arabidopsis type-II ROPs with the plasma membrane requires a conserved C-terminal sequence motif and a proximal polybasic domain

Plant ROPs (or RACs) are soluble Ras-related small GTPases that are attached to cell membranes by virtue of the post-translational lipid modifications of prenylation and S-acylation. ROPs (RACs) are subdivided into two major subgroups called type-I and type-II. Whereas type-I ROPs terminate with a conserved CaaL box and undergo prenylation, type-II ROPs undergo S-acylation on two or three C-terminal cysteines. In the present work we determined the sequence requirement for association of Arabidopsis type-II ROPs with the plasma membrane. We identified a conserved sequence motif, designated the GC-CG box, in which the modified cysteines are flanked by glycines. The GC-CG box cysteines are separated by five to six mostly non-polar residues. Deletion of this sequence or the introduction of mutations that change its nature disrupted the association of ROPs with the membrane. Mutations that changed the GC-CG box glycines to alanines also interfered with membrane association. Deletion of a polybasic domain proximal to the GC-CG box disrupted the plasma membrane association of AtROP10. A green fluorescent protein fusion protein containing the C-terminal 25 residues of AtROP10, including its polybasic domain and GC-CG box, was primarily associated with the plasma membrane but a similar fusion protein lacking the polybasic domain was exclusively localized in the soluble fraction. These data provide evidence for the minimal sequence required for plasma membrane association of type-II ROPs in Arabidopsis and other plant species.     

Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain

Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.     

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.     

Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif

Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser(336), Ser(337), Ser(342), and Ser(349) in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with beta-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous beta-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both beta-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a beta-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified beta-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that beta-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed beta-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in beta-arrestin binding to CCR5.     

Genome-wide analysis of PDZ domain binding reveals inherent functional overlap within the PDZ interaction network

Binding selectivity and cross-reactivity within one of the largest and most abundant interaction domain families, the PDZ family, has long been enigmatic. The complete human PDZ domain complement (the PDZome) consists of 267 domains and we applied here a Bayesian selectivity model to predict hundreds of human PDZ domain interactions, using target sequences of 22,997 non-redundant proteins. Subsequent analysis of these binding scores shows that PDZs can be divided into two genome-wide clusters that coincide well with the division between canonical class 1 and 2 PDZs. Within the class 1 PDZs we observed binding overlap at unprecedented levels, mediated by two residues at positions 1 and 5 of the second α-helix of the binding pocket. Eight PDZ domains were subsequently selected for experimental binding studies and to verify the basics of our predictions. Overall, the PDZ domain class 1 cross-reactivity identified here implies that auxiliary mechanisms must be in place to overcome this inherent functional overlap and to minimize cross-selectivity within the living cell. Indeed, when we superimpose PDZ domain binding affinities with gene ontologies, network topology data and the domain position within a PDZ superfamily protein, functional overlap is minimized and PDZ domains position optimally in the binding space. We therefore propose that PDZ domain selectivity is achieved through cellular context rather than inherent binding specificity.     

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.